The goal of this work is to understand how a peptide encoded by an upstream open reading frame (uORF) controls the movement of ribosomes on mRNA and regulates gene expression. The arginine attenuator peptide (AAP) is encoded by a uORF in mRNAs specifying a fungal arginine (Arg) biosynthetic enzyme and reduces gene expression when Arg is high. AAP-mediated regulation is observed in vitro using extracts from fungi and wheat germ. The nascent AAP causes the ribosome to stall in response to Arg. Stalled ribosomes block access to the downstream start coclon that initiates enzyme synthesis. The AAP is a unique eukaryotic peptide because its synthesis can arrest ribosomes involved in both termination and elongation. Its study provides exceptional opportunities to examine central translational processes that are still not understood. It will provide valuable information on events within the ribosome for evaluating the functions of antibiotics that interfere with translation. Since uORFs that control gene expression are found in animals, plants, fungi, viruses and bacteria, the proposed work is directly relevant to understanding these important regulatory elements.In a working model for regulation, the nascent AAP stalls the ribosome by adopting a conformation that, with Arg, interferes with decoding at the ribosomal A site, or with other steps crucial for translation. The proposed studies will test and refine models for AAP-mediated regulation. Specific aims are: 1. Directly examine the regulatory mechanism by (a) establishing whether stalled ribosomes contain the nascent AAP covalently linked to tRNA to determine whether peptidyl transferase activity is blocked, (b) using photocross-linking of the nascent chain to the ribosome to examine conformational changes in the interaction between the nascent AAP and the ribosome in response to Arg, (c) using photocros s-linking of Arg analogs to determine whether Arg interacts with the AAP or with the translational machinery, and (d) determining whether regulation requires only translational components conserved between prokaryotes and eukaryotes. 2. Examine mutations in nonsense-mediated mRNA decay (NMD) for their effects on mRNA stability and AAP-termination codon recognition. NMD mutations eliminate regulation by the uORF-encoded AAP in vivo. 3. Identify additional trans-acting genes important for translational control by examining mutations in fungi that affect Arg-specific regulation or translation in vivo for their effects on AAP-mediated ribosome stalling in vitro. Cloning and identification of the genes and analysis of their functions should provide a rational explanation of the mutations. 4. Obtain an in-depth understanding of regulatory function by mutational analysis of stalling sites in combination with the use of antibiotics affecting translation and trans-acting mutants affecting regulation.